Báo cáo khoa học: Gonadotropin-releasing hormone and ovarian cancer: a functional and mechanistic overview docx

MINIREVIEW Gonadotropin-releasing hormone and ovarian cancer: a functional and mechanistic overview Wai-Kin So, Jung-Chien Cheng, Song-Ling Poon and Peter C K Leung Department of Obstetrics and Gynecology, University of British Columbia, Vancouver, Canada Keywords apoptosis; G protein; GnRH; gonadotropinreleasing hormone; growth factor; invasion; MAPK; migration; ovarian cancer; proliferation Correspondence P C K Leung, Department of Obstetrics and Gynecology, University of British Columbia, 2H30, 4490 Oak Street, Vancouver, BC, Canada V6H 3V5 Fax: +1 604 875 2717 Tel: +1 604 875 2718 E-mail: peleung@interchange.ubc.ca (Received May 2008, revised August 2008, accepted 15 August 2008) doi:10.1111/j.1742-4658.2008.06679.x The hypothalamic decapeptide gonadotropin-releasing hormone (GnRH) is well known for its role in the control of pituitary gonadotropin secretion, but the hormone and receptor are also expressed in extrapituitary tissues and tumor cells, including epithelial ovarian cancers It is hypothesized that they may function as a local autocrine regulatory system in nonpituitary contexts Numerous studies have demonstrated a direct antiproliferative effect on ovarian cancer cell lines of GnRH and its synthetic analogs This effect appears to be attributable to multiple steps in the GnRH signaling cascade, such as cell cycle arrest at G0 ⁄ G1 In contrast to GnRH signaling in pituitary gonadotropes, the involvement of Gaq, protein kinase C and mitogen-activated protein kinases is less apparent in neoplastic cells Instead, in ovarian cancer cells, GnRH receptors appear to couple to the pertussis toxin-sensitive protein Gai, leading to the activation of protein phosphatase, which in turn interferes with growth factor-induced mitogenic signals Apoptotic involvement is still controversial, although GnRH analogs have been shown to protect cancer cells from doxorubicin-induced apoptosis Recently, data supporting a regulatory role of GnRH analogs in ovarian cancer cell migration ⁄ invasion have started to emerge In this minireview, we summarize the current understanding of the antiproliferative actions of GnRH analogs, as well as the recent observations of GnRH effects on ovarian cancer cell apoptosis and motogenesis The molecular mechanisms that mediate GnRH actions and the clinical applications of GnRH analogs in ovarian cancer patients are also discussed Gonadotropin theory of ovarian cancer Ovarian cancer is the most lethal gynecological malignancy Although epithelial ovarian carcinomas account for approximately 90% of all human ovarian cancers, the etiology of this disease is poorly understood Fathalla proposed the ‘incessant ovulation theory’ in 1971, suggesting that continuous ovulation, associated with successive rounds of surface rupture and repair, increases the chance of accumulating genetic aberrations and therefore malignant transformation [1] The hypothesis is supported by substantive epidemiological data For example, one case–control study of 150 ovarian cancer patients under the age of 50 years demonstrated that the risk of ovarian cancer decreased with increasing numbers of live births, increasing numbers Abbreviations AP-1, activator protein-1; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; GnRH, gonadotropin-releasing hormone; GPCR, G-protein-coupled receptor; IGF-I, insulin-like growth factor-I; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; MMP, matrix metalloproteinase; NF-jB, nuclear factor kappa B; OSE, ovarian surface epithelium; PKC, protein kinase C; PLC, phospholipase C; PP2A, protein phosphatase 2A; PTP, phosphotyrosine phosphatase; PTX, pertussis toxin; TIMP, tissue inhibitor of metalloproteinases 5496 FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS W.-K So et al of incomplete pregnancies, and the use of oral contraceptives [2] Another prevailing hypothesis addressing the development of ovarian cancer was proposed by Cramer and Welch in 1983 Their ‘gonadotropin theory’ proposed that excessive gonadotropin stimulation contributes to ovarian carcinogenesis [3] The risk of ovarian cancer increases during the perimenopausal period, when serum gonadotropin levels peak and thereafter remain elevated [4,5] Moreover, only 10– 15% of tumors appear in premenopausal women [6] Likewise, polycystic ovary syndrome patients (with high luteinizing hormone levels) are more prone to ovarian cancer [7] Epidemiologic evidence supports the idea that pregnancies, breast feeding, and oral contraceptive use, which suppress pituitary gonadotropin secretion, reduce the risk of ovarian cancer [8–11] Experimentally, expression of gonadotropin receptors has been detected in ovarian cancer tissue and in the precursor ovarian surface epithelium (OSE) cells [12– 14] Gonadotropins activate mitogenic pathways, including the extracellular signal-regulated kinase (ERK) pathway [14–16], and promote ovarian cancer cell proliferation [12–14] and invasion [17] Desensitization or downregulation of the gonadotropin-releasing hormone (GnRH) receptors on pituitary gonadotropes by chronic administration of GnRH agonists or competitive binding of the GnRH receptors by GnRH antagonists can block gonadotropin secretion and subsequently suppress gonadotropin-dependent functions in the ovary [18] GnRH agonists have also been shown to inhibit the growth of heterotransplanted ovarian cancer in nude mice, presumably via altering circulating gonadotropin or steroid levels [19], but a direct effect of GnRH on the cancer cells cannot be excluded GnRH ⁄ GnRH receptor autocrine system in ovarian cancer cells Expression of GnRH receptors and specific GnRHbinding sites have been detected in primary cultures of ovarian carcinomas [20] and ovarian carcinoma biopsy specimens [21,22], including mucinous and serous subtypes [23] The widespread presence (> 80%) of GnRH-binding sites in biopsy samples [24,25] supports the involvement of a GnRH regulatory system in ovarian cancers We and others have also demonstrated the presence of GnRH receptors in various established ovarian cancer cell lines, including BG-1, OVCAR-3, SKOV-3, EFO-21 and EFO-27 (Table 1) [20,26–31] The level of GnRH receptor expression in the ovarian cancer cell lines was about 10-fold lower than that in pituitary aT3 cells [20] GnRH and ovarian cancer The extremely short half-life of hypothalamic GnRH makes it an unlikely candidate to act on the ovary via the systemic circulation and suggests the existence of a local source of GnRH in ovarian cancer cells Indeed, our group and others have detected GnRH-I mRNA in normal OSE and immortalized OSE cells, as well as in primary cultures of ovarian tumors and ovarian carcinoma cell lines such as EFO-21, EFO-27, CaOV-3, OVACR-3 and SKOV-3 [32,33] Similarly, GnRH-II mRNA has been detected in normal and neoplastic OSE cell lines and primary cultures of ovarian carcinomas [28] GnRH-like immunoreactivity was detected in conditioned media [34] and cell lysates [21] from ovarian cancer cell lines The latter possessed bioactivity comparable to that of authentic GnRH, as it stimulated luteinizing hormone release from rat pituitary [21] Incubation of ES-2 ovarian cancer cells in vitro with a GnRH-I antibody inhibited cell proliferation in a time- and dose-dependent manner [34], whereas Emons reported a significant increase in EFO-21 and EFO-27 ovarian cancer cell proliferation after GnRH-I antiserum treatment [35] Despite this discrepancy, these studies provide direct evidence for the endogenous secretion of bioactive GnRH as an autocrine growth-regulatory loop in ovarian cancer cells Our laboratory demonstrated the existence of an autocrine loop involving GnRH and the GnRH receptor in primary cultures of human OSE cells (scraped from the ovarian surface during laparoscopies for nonmalignant disorders) [32] The GnRH agonist [D-Trp6]GnRH had a direct inhibitory effect on growth of OSE cells in a time- and dose-dependent manner This inhibitory effect was reversed by cotreatment with the GnRH receptor antagonist antide [32] Moreover, the GnRH agonist has a homologous regulatory effect on the expression of GnRH and the GnRH receptor in OSE cells, which further supports the presence of an autocrine regulatory GnRH system that is operational in the ovary Antiproliferative effect of GnRH analogs on ovarian cancer Numerous in vitro studies have reported a growthmodulating effect of GnRH-I ⁄ GnRH-II and their synthetic analogs in various GnRH receptor-bearing ovarian cancer cell lines (Table 1) In most cases, GnRH-I and its agonists were reported to inhibit ovarian cancer cell proliferation, as judged by decreased cell number or DNA synthesis For example, our laboratory has reported that treatment with the agonist [D-Ala6]GnRH caused a time- and dose-dependent inhibition of cell proliferation in the ovarian cancer FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS 5497 5498 10)11–10)5 GnRH-I agonist [D-Ala6]GnRH GnRH-I agonist [D-Ala6]GnRH Native GnRH-I GnRH-I agonist triptorelin GnRH-I agonist triptorelin GnRH-I agonist [D-Ala6]GnRH GnRH-I antagonist cetrorelix GnRH-I agonist triptorelin GnRH-I agonist triptorelin Native GnRH-II [32] [36] [35] [53] [34] [28] [46] [43] [37] [27] GnRH-I agonist triptorelin GnRH-I antagonist cetrorelix GnRH-II 10)5 GnRH-I agonist triptorelin GnRH-I antagonist cetrorelix, Hoe-013 Human OSE OVCAR-3 + + + + + SKOV-3 EFO-21 EFO-27 + + + M 10)12–10)5 + + R-I OVCAR-3 OV-1063 Cell line M 10)9–10)7 10)11–10)5 10)11–10)5 10)11–10)5 M M M M M + + ) + + + + ) EFO-27 EFO-21 OVCAR-3 SKOV-3 EFO-21 BG-1 OVCAR-3 EFO-27 SKOV-3 + + EFO-21 + + IOSE29, IOSE28-EC HTOA M 10)9–10)7 10)9–10)5 M EFO-27 ES-2 M 10, 1000 ngỈmL)1 10)7 + + EFO-21 EFO-27 EFO-21 M M M 10)11–10)7 10)11–10)7 10)5 M 10)11–10)5 M M M [30] [44] 10)8–10)5 10)8–10)5 10)7–10)5 GnRH-I antagonist cetrorelix GnRH-I agonist triptorelin GnRH-I agonist triptorelin [31] Dose GnRH analogs Reference + + + + + ) + ) + + ND ND ) ND + ) + ND ND ND + ) ND ND R-II Triptorelin: antiproliferation Cetrorelix: antiproliferation GnRH-II: antiproliferation Triptorelin: antiproliferation for EFO-21, but not for SKOV-3 GnRH-II: antiproliferation for all cell lines Increase ⁄ decrease cell proliferation (10 ngỈmL)1), antiproliferation (1000 ngỈmL)1) Antiproliferation Antiproliferation and cell cycle arrest (10)9, 10)5 M), apoptosis (10)5 M) Antiproliferation and cell cycle arrest Protection against DOX-induced apoptosis Antiproliferation Antiproliferation for EFO-21 but not EFO-27 Antiproliferation Antiproliferation and DNA fragmentation Antiproliferation Antiproliferation and cell cycle arrest Antiproliferation Action Table In vitro effects of GnRH-I and GnRH-II analogs on OSE and ovarian cancer cell lines R-I, GnRH-I receptor; R-II, GnRH-II receptor; ND, not determined; DOX, doxorubicin; +, positive; ), negative Expression of GnRH receptors is based on reports published by individual groups GnRH and ovarian cancer W.-K So et al FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS GnRH-II agonist D-Arg(6)-Azagly(10)-NH2 GnRH-I agonist leuprolide GnRH-I antagonist cetrorelix GnRH-II agonist D-Arg(6)-Azagly(10)-NH2 GnRH-I agonist triptorelin GnRH-II agonist D-Arg(6)-Azagly(10)-NH2 GnRH-I agonist [D-Ala6]GnRH GnRH-II antagonists: [Ac-D2Nal1, D-4Cpa2, D-3Pal3, D-Lys6, D-Ala10]GnRH-II, [Ac-D2Nal1, D-4Cpa2, D-3Pal3, D-Lys6, Leu8, D-Ala10]GnRH-II, [Ac-D2Nal1, D-4Cpa2, D-3Pal3,6, Leu8, D-Ala10]GnRH-II GnRH-I agonist triptorelin [57] [54] [50] [42] [41] [58] [64] GnRH-II agonist D-Arg(6)-Azagly(10)-NH2 GnRH-I agonist triptorelin [127] [63] GnRH analogs Reference Table (Continued) 10)10–10)7 M M M + + ) + OVCAR-3 SKOV-3 OVCAR-3 10)5 10)7 M 10)7 M 10)7 M 10)10–10)8 SKOV-3 + + + + + + + + + M SKOV-3 CaOV-3 SKOV-3 OVCAR-3 OVCAR-3 SKOV-3 CaOV-3 OVCAR-3 EFO-21 10)8–10)6 M M + ) + + OVCAR-3 SKOV-3 OVCAR-3 CaOV-3 10)7 10)6 + EFO-21 M 10)11–10)5 R-I Cell line Dose ND + + ND ND ND ND + ND ND ND ND + + + ND ND + R-II Induction of OVCAR-3 invasion (10)10–10)8 M), suppression of SKOV-3 invasion at 10)8 and 10)7 M Apoptosis Cell migration and invasion Antiproliferation Antiproliferation Apoptosis Antiproliferation and apoptosis Protection against DOX-induced apoptosis Suppression of 17b-estradiol-induced EFO-21 and OVCAR-3 cell proliferation, but no effect on SKOV-3 Action W.-K So et al GnRH and ovarian cancer 5499 GnRH and ovarian cancer W.-K So et al Table Overview of trials using GnRH agonists in ovarian cancer CR, complete response; PR, partial response; SD, stable disease Reference Drug [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] [117] [118] [106] [119] [120] Leuprolide Leuprolide Leuprolide Leuprolide Leuprolide Leuprolide Leuprolide Triptorelin Triptorelin Triptorelin Triptorelin Triptorelin Triptorelin Goserelin Goserelin Patients acetate acetate acetate acetate acetate acetate acetate CR PR SD 18 25 32 32 37 12 41 19 20 40 14 68 23 30 0 0 0 0 0 0 4 11 0 0 2 15 4 14 11 cell line OVCAR-3 Significant inhibition was detected as early as days after treatment [36] Interestingly, GnRH-I antagonists consistently act like agonists and inhibit cell proliferation in various cell lines (Table 2) GnRH-I antagonists were reported to be more potent than equimolar concentrations of agonists in inhibiting ovarian cancer cell growth [29,37] This phenomenon was also observed in endometrial [38], prostate [39] and breast cancers [40], suggesting that the dichotomy between GnRH-I agonists and antagonists in the pituitary might not be applicable to cancer cells The exact mechanism underlying the difference between the pituitary and extrapituitary tissues remains to be elucidated In contrast to the situation with GnRH-I, functional studies on GnRH-II are rather limited We and others have reported that, like GnRH-I agonists, GnRH-II and its agonists, such as d-Arg(6)-Azagly(10)-NH2, exert an antiproliferative effect on ovarian cancer cell lines [41,42] The antiproliferative effect of GnRH-II is more potent than that of GnRH-I [37] Interestingly, it has been reported that a GnRH-II agonist inhibits the growth of SKOV-3 cells, which are GnRH-I receptor-negative and unresponsive to GnRH-I [27] The antiproliferative effect of GnRH-I is associated with an induction of cell cycle arrest at G0 ⁄ G1 [43–46], coupled with a downregulation of Cdk expression [44] and cyclin A–Cdk2 complex levels [46], or inhibition of telomerase activity without alteration of RNA expression [47] In addition to cell cycle arrest, apoptosis may also be involved in the antiproliferative action of GnRH GnRH-I agonists have been reported to induce prostate cancer apoptosis [48] In ovarian cancer cells, a 5500 high concentration (10)5 m) of GnRH agonist has been reported to induce tumor necrosis factor-a secretion, interchromosomal DNA fragmentation, and a marginal apoptotic effect [49] An equimolar concentration of the GnRH-I antagonist cetrorelix induced apoptosis by upregulating p53 and p21 protein levels, whereas concentrations as low as 10)9 m resulted in antiproliferative effects [46] Recently, apoptosis was shown to be induced by a low concentration of cetrorelix in ovarian cancers [50] We also observed DNA fragmentation after prolonged (6 days) low-dose GnRH-I agonist treatment [36] In most studies, however, apoptosis was induced only when ovarian cancer cells were treated with GnRH-I analogs at relatively high concentrations or for a prolonged time Although Fas and FasL were detected in the majority of ovarian carcinomas and ovarian cancer cell lines [51,52], and GnRH agonists such as buserelin dose-dependently induced FasL expression in ovarian cancer cells [52], a causative linkage between Fas ⁄ FasL and the antiproliferative action of GnRH has not been established Indeed, there is no consensus about the proapoptotic role of GnRH The antiproliferative effect of GnRH has been mainly attributed to the cytostatic action of GnRH rather than induction of apoptosis GnRH-I agonists, including triptorelin [44,53] and leuprolide [54], were marginally effective or ineffective in inducing ovarian cancer cell apoptosis In contrast, these agonists exerted a protective effect against the cytotoxic action of the chemotherapy drug doxorubicin [53,54] Abolition of GnRH action by GnRH receptor knock-down increased doxorubicin-induced apoptosis [55] A GnRH-generated protective effect against doxorubicin-induced apoptosis was also observed in human granulosa, breast cancer and endometrial cancer cells [55,56] The underlying mechanism of this protective effect is unknown, although activation of nuclear factor kappa B (NF-jB) may be involved Triptorelin was shown to activate NF-jB in ovarian cancer cells, and blockage of NF-jB translocation into the nucleus reversed GnRH-induced protection against doxorubicin [53] (Fig 1F) In this case, the antitumor (antiproliferative) and antiapoptotic effects of GnRH would appear to be paradoxical, but in fact doxorubicin and most chemotherapy drugs are more efficacious towards rapidly dividing cells, and thus the cell cycle arrest induced by GnRH can protect the tumor cell from doxorubicin Regarding GnRH-II, we and others showed that GnRH-II and its antagonist induced ovarian cancer cell apoptosis [57,58], which was mediated by p38 mitogen-activated protein kinase (MAPK) and caspase-3 activation [57,58] Furthermore, an antitumor effect of GnRH-II antagonists was FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS W.-K So et al GnRH and ovarian cancer GnRH-I agonist/ antagonist GnRH-I antagonist GnRH-I receptor GnRH-II receptor ? Gαi Gαi G FK E AC cAMP demonstrated in nude mice bearing ovarian cancer cell xenografts [58] In contrast to the relatively large number of studies on GnRH actions such as antiproliferative and apoptotic ⁄ antiapoptotic effects, reports of GnRH influences on other parameters of ovarian cancer progression, such as tumorigenic or metastatic processes, are limited Spread of ovarian cancer beyond the ovaries to the peritoneal cavity leads to later staging of the disease and poor prognosis The fact that a high proportion of advanced-stage (stages III and IV) ovarian carcinomas express GnRH receptor mRNA and protein, as compared to early-stage carcinomas [59], has prompted us to investigate the participation of GnRH in regulating migration and invasion in ovarian cancer Previously, we have demonstrated the potency of GnRH-I and GnRH-II regulation of the urokinasetype plasminogen activator ⁄ plasminogen inhibitor system and matrix metalloproteinase (MMP)-2, MMP9, and tissue inhibitor of metalloproteinases (TIMP)-1 in other gynecological tissues, including extravillous cytotrophoblasts and decidual stromal cells [60–62] These proteolytic enzymes are involved in the degradation and remodeling of extracellular matrix, which has been implicated in the multistep process of metastasis formation By activating MMP-2 and MMP-9 promoters to increase gene expression, GnRH agonists PTP B D C EGFR A β γ F NFκB Translocation Fig GnRH-I signaling in ovarian cancer cells (A) Through Gai, GnRH-I analogs activate PTP to dephosphorylate EGFR and abolish EGF-induced ERK activation, c-fos expression and proliferation (B) Gbc subunit activates ERK and mediates GnRH-I-induced growth inhibition (C) GnRH-I activates ERK through a PKC-dependent or PKC-independent pathway to inhibit proliferation (D) GnRH-I activates JNK, which increases AP-1 activity and JunD–DNA binding to extend the cell cycle (E) GnRH-I suppresses apoptosis through activation of PP2A (F) GnRH-I stimulates NF-jB activity and nuclear translocation to protect ovarian cancer cells from apoptosis (G) GnRH-I acts through Gai to counteract forskolin (FK)-induced cAMP The presence of a functional GnRH-II receptor has yet to be evaluated Dashed arrows represent the EGF-stimulated mitogenic signaling pathway; FS, forskolin; AC; adenylate cyclase EGF P P Sos Shc P PKC PP2A JNK JunD NFκB MEK ERK1/2 EGFR Expression AP-1 c-fos expression Apoptosis Cell cycle Proliferation stimulated the migration and invasion potential of CaOV-3 and OVCAR-3 ovarian cancer cells The GnRH-induced increase in invasiveness and migratory activity was blocked by neutralizing antibodies against MMP-2 and MMP-9 [63] This motogenic action of GnRH was mediated by GnRH receptor and c-Jun N-terminal kinase (JNK), but not by ERK or p38 MAPK [63] (Fig 2C) We further investigated the effect of GnRH-II in ovarian cancer cells The GnRH-II agonist d-Arg(6)-Azagly(10)-GnRH-II, like GnRH-I agonists, stimulated OVCAR-3 cell invasion Interestingly, high doses of GnRH-I and GnRH-II agonists were observed to reduce the invasive potential of SKOV-3 cells by altering the balance between MMP and TIMP [64] In this regard, it is noteworthy that GnRH-I agonists and antagonists have been reported to inhibit the migration and invasion of prostate cancer, breast cancer and epidermoid carcinoma cells [65–67] Also, breast cancer cell invasiveness was suppressed in vitro by both GnRH-I and GnRH-II [67] Signaling and mechanism of GnRH action in ovarian cancer cells As a member of the serpentine receptor family, the GnRH receptor transmits its signals mainly through heterotrimeric G-proteins (GTP-binding proteins) FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS 5501 GnRH and ovarian cancer W.-K So et al EGF GnRH-II receptor ? GnRHII EGFR GnRH-I receptor A Gαi D C p38 P PTP JNK B PKC P P Shc Sos MEK AP-1 ERK1/2 Migration/ invasion Elk-1 c-fos expression Apoptosis Proliferation Upon stimulation, Ga dissociates from the Gbc dimer and changes to its active GTP-bound form According to the subtype of their a-subunits, G-proteins can be categorized into four groups: Gs, Gi, Gq ⁄ 11, and G12 ⁄ 13 Gas and Gai mainly exert their effects via stimulating or inhibiting, respectively, adenylate cyclase to modulate the production of cAMP Gaq activates membrane-associated phospholipase C (PLC), which hydrolyzes phosphoinositides to generate the second messengers inositol 1,4,5-triphosphate and diacylglycerol, resulting in intracellular Ca2+ mobilization and protein kinase C (PKC) activation Moreover, Gaqactivated PKC can activate MAPKs, including JNK, ERK and p38 MAPK [68,69] It is well established that the GnRH receptor interacts with multiple G-proteins, and that specificity is cell context dependent [68] In hypothalamic neurons, the GnRH receptor interacts with Gaq, Gas and Gai [70] In pituitary gonadotropes, GnRH preferentially or exclusively stimulates Gaq [71] However, Gai has been shown to mediate GnRH receptor signaling in tumor cells such as ovarian cancer [50,72,73], endometrial cancer [72,73] and prostate cancer [74] cells Consequently, the downstream signaling pathways and the physiological outcomes of GnRH action may be quite different in the gonadotropes and extrapituitary tissues The mechanism that leads to the inconsistency of GnRH signaling and biological outcome in different tissues is unknown It is unlikely that divergent signaling is due to alternatively spliced variants or mutant 5502 Fig GnRH-II signaling in ovarian cancer cells (A) Similar to GnRH-I, GnRH-II activates PTP to inhibit EGFinduced ERK activation, c-fos expression and proliferation (B) Through PKC, GnRH-II activates ERK and Elk-1 to suppress proliferation (C) GnRH-II activates JNK to induce migration ⁄ invasion (D) GnRH-II activates Gai or p38 and AP-1 to induce apoptosis Dashed arrows represent the EGF-stimulated mitogenic signaling pathway receptors in cancer cells, as expression of the wild-type GnRH receptor has been confirmed in OVCAR-3, EFO-21 and EFO-27 ovarian cancer cell lines and human OSE cells [32,72] On the other hand, the receptor may oscillate, in a cell context-dependent manner, between multiple conformations, each with specific ligand and intracellular signaling complex selectivity According to this hypothesis, the receptor can adopt a conformation that preferentially binds certain ligand(s), in response to different ligand concentrations The intracellular signaling complex could in turn stabilize the receptor conformation and favor the binding of the ligand [75] There is direct evidence supporting the existence of multiple conformations of a G-protein-coupled receptor [76] Thus, the GnRH receptor in gonadotropes may preferentially bind GnRH-I and be coupled to the Gaq–PLC pathway in order to modulate gonadotropin expression and secretion, whereas in cancer cells, the GnRH receptor coupling to Gai is selectively recognized and activated by GnRH-II in order to regulate cell proliferation and apoptosis This kind of conformational preference may be a result of cell context, including cell type and prior exposure to other hormones [75] Concrete evidence for this specificity has been generated for the Xenopus GnRH receptor: activation of PKC, which phosphorylates the C-terminus of the receptor, led to a marked increase in GnRH-II binding to the Xenopus GnRH-I receptor, but had no effect on GnRH-I binding [75] This theory could resolve questions regarding the FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS W.-K So et al GnRH system in different tissues, including why a lower binding affinity of GnRH agonists has been observed in tumor cells than in pituitary gonadotropes [75], why GnRH antagonists act like agonists in cancer cells (Table 2), and why GnRH-II is observed to be more potent than GnRH-I in inhibiting cancer cell proliferation through Gai, but less potent in stimulating Gaq-mediated gonadotropin secretion in pituitary gonadotropes [37,77] At present, the identity of the GnRH receptor(s) that mediate the antiproliferative actions of GnRH in tumor cells and the agonistic effects of GnRH antagonists in cancer cells is still controversial Grundker and co-workers have compared the GnRH responsiveness of SKOV-3 and EFO-27 ovarian cancer cells, and proposed that their responsiveness correlates with the expression of GnRH receptors Accordingly, EFO-27 cells expressing GnRH-I receptor but not GnRH-II receptor responded to the GnRH-I agonist triptorelin but not to antagonists, even at high concentrations (10)5 m) [27,30,78] By contrast, in SKOV-3 cells, which are reportedly GnRH-I receptor-negative but GnRH-II receptor-positive, both the GnRH-I antagonist cetrorelix and GnRH-II, but not triptorelin, inhibited cell growth [27,37] or EGF-induced c-fos expression [78] Cell lines expressing both GnRH-I and GnRH-II receptors were responsive to all of the treatments These findings were in accordance with other reports that growth of GnRH-II receptorexpressing Ishikawa and Hec-IA endometrial cancer cell lines was inhibited by a GnRH-I antagonist [27,38] Moreover, the GnRH-I agonist-induced antiproliferative effect was abolished by GnRH-I receptor knock-out, whereas the antiproliferative effects induced by the antagonist cetrorelix or GnRH-II agonist persisted [27,79] These results support the hypothesis that the GnRH-I receptor mediates the actions of GnRH agonists, whereas GnRH antagonists and GnRH-II may act through an additional receptor, i.e the putative GnRH-II receptor Although a functional GnRH-II receptor has not yet been identified in humans, the presence and ⁄ or functionality of a type II receptor has not been ruled out The human GnRH-I antagonist 135-18 and GnRH-II are capable of activating the marmoset GnRH-II receptor, which is overexpressed in COS-7 cells [80] However, the presence of only one class of specific, high-affinity ⁄ low-capacity receptors in humans has not been sufficiently demonstrated Binding of the agonist [D-Trp6]GnRH was displaced by the antagonist SB-75, suggesting that both analogs bind to the same receptor on OV-1063 cells [29] Thus, the precise mechanisms underlying the divergence of signaling pathways and biological GnRH and ovarian cancer actions in pituitary gonadotropes and tumor cells remain to be determined Gai and ⁄ or Gaq have been detected in GnRH-I receptor-expressing ovarian cancer cell lines and surgically removed ovarian carcinomas [72,73] GnRH-I receptor has been shown, by disuccinimidyl suberate cross-linking experiments, to interact physically with Gai and Gaq [72] Functionally, it has been suggested that GnRH-I receptor couples with Gai, which is common in tumor cells [50,72–74] Gai is pertussis toxin (PTX)-sensitive and is not affected by cholera toxin PTX induced ADP-ribosylation of the a-subunit in the GnRH-I receptor-positive tumor cell membrane [50,72–74] and thus impaired GnRH-I receptor-linked cellular events, including GnRH-induced phosphatase activity [50,72,73], apoptosis [50], and antiproliferative actions [72,74] Conversely, incubation with GnRH agonists substantially antagonized the PTX-catalyzed ADP-ribosylation of Gai [72–74] Furthermore, Gai significantly counteracted the forskolin-induced increase in intracellular cAMP levels [74] (Fig 1G) These findings strongly indicate that Gai is the major G-protein mediating GnRH actions in tumor cells In addition to the PTX-sensitive Gai, G-protein bc-subunits also mediated GnRH agonist-induced antiproliferative effects and ERK activation in ovarian cancer cell lines, and such ERK activation was blocked by ectopic expression of the C-terminus of a-adrenergic receptor kinase I, an antagonist of G-protein beta gammasubunits [81] (Fig 1B) In pituitary gonadotropes, PKC and PLC act downstream of Gaq to relay the GnRH receptor signals However, the roles of PKC and PLC in GnRH receptor signal transduction in tumor cells are less clear There is evidence that the signaling pathways induced by GnRH-I in pituitary gonadotropes, including PLC and PKC, are not activated by the GnRH-I agonist triptorelin in ovarian, endometrial and breast cancer cell lines [35,36] Similarly, GnRH-I-stimulated MAPK activation in pituitary aT3 cells was abolished by 4b-phorbol 12-myristate 13-acetate pretreatment (to deplete PKC) or by depletion of Ca2+, whereas the GnRH-I agonist activated MAPK via a PKC-independent mechanism to inhibit growth of CaOV-3 ovarian cancer cells [81] However, evidence supporting PKC involvement in GnRH actions in tumor cells or extrapituitary tissues is available Cetrorelix stimulated PKC activity in DU-145 prostate cancer cells, resulting in an increase in phosphorylation of the PKC substrate MARCKS [82] By activating PKC, GnRH analogs inhibited the EGF receptor (EGFR) signal and the growth of prostate cancer xenografts in athymic mice [83] and cell invasion in vitro [82] Essentially, prostate FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS 5503 GnRH and ovarian cancer W.-K So et al cancer cells carrying a mutated EGFR that lacks the target site for PKC are resistant to GnRH-induced in vivo and in vitro growth inhibition [82,83] Our laboratory has recently demonstrated that 12-O-tetradecanoyl phorbol-13-acetate (a PKC-activating phorbol ester) can mimic the effects of GnRH-I and GnRH-II in stimulating ERK1 ⁄ phosphorylation and antiproliferation in ovarian cancer cells Furthermore, the effects of GnRH were abolished by pretreatment with the PKC inhibitor GF109203X [41] (Figs 1C and 2B) By analogy, the PKC inhibitor calphostin C and the activator 12-O-tetradecanoyl phorbol-13-acetate can block and mimic, respectively, the antiproliferative action of the GnRH agonist buserelin on surgically removed uterine leiomyoma [84] cells In human granulosa-luteal cells, a GnRH agonist stimulates MAPK activation through a PKC-dependent pathway [85] However, the direct effect on PKC activation and the identity of the PKC isoform activated by GnRH are still under investigation In pituitary gonadotrope cells, there is ample evidence that GnRH-I activates MAPK members, including ERK, JNK and p38, in a PKC-dependent pathway to control gonadotropin secretion [68] MAPK members mediate distinct roles in GnRH-induced gonadotropin subunit gene transcription [86–88] In sharp contrast to the situation with pituitary gonadotropes, our understanding of MAPK activation by GnRH analogs in cancer cells is rather limited In ovarian cancer OVCAR-3 cells and placental cancer JEG-3 cells, we have demonstrated biphasic activation of ERK by [D-Ala6]GnRH High doses of GnRH agonist (10)7 and 10)6 m) significantly activated ERK, whereas a low dose (10)10 m) resulted in decreased ERK activation [89] In another study, phosphorylation of ERK, Sos and Shc was induced by the GnRH-I agonist leuprolide [81] Leuprolide-induced ERK activation was rapid (within min) and long-lasting (sustained up to 24 h) ERK appeared to mediate the antiproliferative action of GnRH and such growth inhibition could be reversed by the mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 [81] The dual roles of ERK in mediating mitogenic effects (by growth factors) and antiproliferative effects (by GnRH) seem contradictory Paradoxical actions of ERK have also been reported in PC-12 cells: transient activation induced by EGF led to proliferation, and nerve growth factor-induced prolonged ERK activation caused differentiation and cessation of proliferation [90] It has been suggested that the duration of ERK activation is important in determining its actions and thus the resultant cell fate [90] In addition to ERK, JNK was activated by triptorelin through induction of c-jun mRNA expression and 5504 protein phosphorylation [91], in a manner that was independent of PLC and PKC [92] The same group further demonstrated that triptorelin treatment increased activator protein-1 (AP-1) activity and JunD–DNA binding, and extended the cell cycle [43] (Fig 1D) As JNK and c-jun are implicated in cell cycle regulation [93], it is logical to hypothesize that the JNK–c-jun–AP-1 pathway mediates the GnRHinduced antiproliferative effect This pathway may act in concert with NF-jB, as it was shown to protect tumor cells from doxorubicin-induced apoptosis in the same system JunD is proposed to act as a modulator of cell proliferation and to cooperate with the antiapoptotic and antiproliferative functions of GnRH However, further investigation is necessary to resolve the observations by others that the GnRH-I agonist leuprolide and a GnRH-II agonist could not activate JNK [42,81], and that p38 was implicated in GnRH-IIinduced ovarian cancer cell apoptosis [57] (Fig 2D) In addition to the direct antiproliferative signal elicited through PKC-dependent or PKC-independent pathways, the antiproliferative effect on tumor cells of GnRH may arise from its ability to activate phosphatases and counteract the mitogenic signals induced by growth factors [94] (Fig 1A) In OSE and epithelial ovarian carcinomas, EGF and various growth factors are secreted and function locally to promote tumor proliferation and progression [95] Binding of growth factors to their cognate receptor tyrosine kinases induces receptor dimerization and autophosphorylation Phosphorylated receptor tyrosine kinases phosphorylate adaptor and effector molecules to subsequently initiate a phosphorylation cascade that is important for the growth-promoting and tumorigenic functions of growth factors Propagation of the phosphorylation cascade and its physiological effects can be terminated by protein phosphatases It has been shown that EGFR, when stimulated by EGF, may phosphorylate itself and other cellular substrates, including Src, in human pancreatic cells; cotreatment with [D-Trp6]GnRH reversed the effect of EGF and led to the dephosphorylation of these proteins [96] In the plasma membrane of tumor cells, phosphotyrosine phosphatase (PTP) or serine ⁄ threonine protein phosphatase 2A (PP2A) were shown to be activated by GnRH agonists [50,54,72,97–100], suggesting that GnRH-I increased the turnover rate of protein phosphorylation ⁄ dephosphorylation and that EGFR is a target of the dephosphorylation activity [72,96] As a result, EGFR phosphorylation [72] and the downstream signaling and mitogenic effects of EGF were abrogated, including EGF-induced MAPK activation [94], immediate early gene c-fos expression [78] and FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS W.-K So et al proliferation [92] Downregulation of receptors for EGF and ⁄ or insulin-like growth factor-I (IGF-I) by GnRH antagonist [101] and in ovarian cancer-xenografted nude mice have been reported [31,102] Interference of growth factor signaling by GnRH analogs has also been demonstrated in prostate cancer cells GnRH-I analogs abrogated the mitogenic effects of EGF and IGF-I and inhibited prostate cancer growth [103,104] by reducing expression of their receptors [103–105], as well as by inhibiting EGF- and IGF-Iinduced receptor phosphorylation [103,104] and c-fos expression GnRH-II agonist was reported to act in a similar fashion, i.e enhancing PTP activity and thus reducing EGF-induced EGFR phosphorylation, MAPK activation and c-fos expression [79] (Fig 2A) Moreover, GnRH-activated phosphatase activity has also been implicated in its antiapoptotic function Doxorubicin decreased the activity of a crucial phosphatase in apoptosis control (PP2A), and induced ovarian cancer cell apoptosis Cotreatment with the GnRH-I agonist leuprolide partially restored PP2A activity and antagonized doxorubicin-induced apoptosis [54] (Fig 1E) Clinical studies on GnRH agonists and antagonists in ovarian cancer In a limited number of studies, GnRH-I agonists have been evaluated for their potential as second-line therapy in patients with refractory and recurrent ovarian cancer who had failed at least one chemotherapy regimen In 2001, the European Organization of Research and Treatment of Cancer Gynecological Cancer Cooperative Group completed the largest reported series of GnRH agonist trials Seventy-four patients with progressive ovarian cancer who had previously undergone platinum-based therapy were treated with the GnRH agonist triptorelin No objective responders were observed Eleven of 68 evaluable patients (16%) had stable disease The median progression-free survival was months in patients with disease stabilization and months for all evaluable patients The median survival for patients with disease stabilization was 17 months, whereas for all patients it was months This study showed that treatment with the GnRH agonist triptorelin has only modest efficacy in patients pretreated with platinum-containing chemotherapy [106] Table summarizes 15 clinical trials, beginning as early as 1988, that have used three different GnRH agonists (leuprolide acetate, triptorelin, goserelin) on relapsed platinum-resistant ovarian cancer patients (Table 2) [106–120] The majority of these trials involved only a limited number of patients GnRH and ovarian cancer Three trials have been completed that compared the use of platinum-based chemotherapy alone or in combination with a GnRH agonist as first-line therapy for ovarian cancer [121–123] A prospective randomized double-blind trial enrolled 135 patients with stage III or IV epithelial ovarian carcinoma, and showed that suppression of endogenous gonadotropins by conventional doses of the GnRH agonist triptorelin produces no relevant beneficial effects in patients with advanced ovarian carcinoma who receive standard surgical cytoreduction and standard platinum-based chemotherapy [121] In the other two studies, patients received carboplatin-containing polychemotherapy and cisplatin alone or chemotherapy plus triptorelin, but no significant differences were seen in terms of response, survival and time to progression [122,123] The ineffectiveness of the GnRH agonist in combination with chemotherapy is postulated to be due to the neutralization of its direct antiproliferative effects by its antiapoptotic activity, as demonstrated by the in vitro data [53,54,124] In vitro data demonstrated that antagonists provided a greater inhibitory effect on ovarian cancer proliferation than agonists [29] Clinically, as GnRH-I antagonists not possess intrinsic gonadotropic activity, the initial ‘flare-up’ phenomenon, which is common in agonist treatment, can be avoided This makes antagonists better tolerated and capable of blocking gonadotropin secretion within a short time frame [125] A clinical trial of the GnRH antagonist cetrorelix was conducted on 17 patients All of the patients had relapsed disease after standard chemotherapy before entering into the trial Three patients (18%) experienced a partial remission with cetrorelix treatment that lasted 2, and months, and six women (35%) had disease stabilization for 1–12 months The median survival was 17 months [126] Conclusions and future prospects To date, our understanding of the GnRH system in tumor cells is still far from complete, especially with regard to the newly identified GnRH-II isoform and the ‘putative’ GnRH-II receptor Although there are suggestive data supporting the existence of a functional mammalian GnRH-II receptor and a role of the GnRH-II receptor in mediating the antiproliferative effects of GnRH-I antagonists and GnRH-II, direct evidence for a functional human GnRH-II receptor and the details of its downstream signaling mechanism are certainly of great physiological importance and research interest The superior antiproliferative effects of GnRH-II as compared to GnRH-I make GnRH-II an attractive target for investigation, and the hormonal FEBS Journal 275 (2008) 5496–5511 ª 2008 The Authors Journal compilation ª 2008 FEBS 5505 GnRH and ovarian cancer W.-K So et al regulation of GnRH-II expression in ovarian cancer cells is presently under investigation in our laboratory The widespread expression of the GnRH receptor in ovarian carcinomas and the well-documented in vitro effects, such as antiproliferation and apoptosis, strongly support the candidacy of GnRH as a promising therapeutic approach for ovarian cancer Elucidation of the efficacy and modes of actions of GnRH-I and GnRH-II, as well as their interactions with growth factors that are known to be important in ovarian cancer progression, is undoubtedly warranted Acknowledgements P.C.K.L is the recipient of a Child & Family Research Institute Distinguished Scholar Award W.K.S., J.C.C and S.L.P were recipients of graduate studentship awards from The Interdisciplinary Women’s 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Ohmichi M, Tasaka K, Kimura A, Kanda Y, Hayakawa J, Tahara M, Hisamoto K, Kurachi H & Murata Y (2000) Activation of the luteinizing hormone beta promoter by gonadotropin-releasing hormone requires... A, Ohmichi M, Kurachi H, Ikegami H, Hayakawa J, Tasaka K, Kanda Y, Nishio Y, Jikihara H, Matsuura N et al (1999) Role of mitogen-activated protein kinase ⁄ extracellular signal-regulated kinase... disease and poor prognosis The fact that a high proportion of advanced-stage (stages III and IV) ovarian carcinomas express GnRH receptor mRNA and protein, as compared to early-stage carcinomas